Methods of treating pulmonary disorders with liposomal amikacin formulations

ABSTRACT

Disclosed herein are methods of treating pulmonary disorders comprising administering to the patient an effective dose of a nebulized liposomal amikacin formulation for at least one treatment cycle, wherein: the treatment cycle comprises an administration period of 15 to 75 days, followed by an off period of 15 to 75 days; and the effective dose comprises 100 to 2500 mg of amikacin daily during the administration period.

RELATED APPLICATIONS

This application is a continuation in part of International ApplicationNo. PCT/US08/062868, filed on May 7, 2008, which claims the benefit ofpriority to U.S. Provisional Application No. 60/916,342, filed on May 7,2007, both of which are hereby incorporated by reference in theirentirety.

BACKGROUND OF THE INVENTION

Cystic fibrosis (CF), also called mucoviscidosis, is an autosomal,recessive, hereditary disease of the exocrine glands. It affects thelungs, sweat glands and the digestive system, causing chronicrespiratory and digestive problems. It is caused by mutations in thecystic fibrosis transmembrane conductance regulator (CFTR) protein. Itis the most common fatal autosomal recessive diseases amongstCaucasions.

The first manifestation of CF is sometimes meconium ileus, occurring in16% of infants who develop CF. Other symptoms of CF manifest duringearly childhood. Both lungs and pancreas produce abnormally viscousmucus. This mucus begins to build up and starts to clog the opening tothe pancreas and the lungs. Pulmonary problems start from the constantpresence of thick, sticky mucus and are one of the most seriouscomplications of CF. The mucus in the lungs can become a growth mediumfor bacteria, resulting in chronic respiratory infections and eventualpermanent damage to the lung tissue. During the end stage of CF, thepatient experiences increased chest congestion, activity intolerance,increased crackles, and increased cough, which often contains sputummixed with blood (hemoptysis) due to the bronchiole bleeding from thelung arteries. A chronic and loose sounding cough is common in peoplewith CF. These thick secretions also obstruct the pancreas, preventingdigestive enzymes from reaching the intestines to help break down andabsorb food. Frequent and foul smelling stools are often an early signof CF along with fatty oil that is visible in the stool. This cancompromise growth and overall nutrition if proper treatment to aiddigestion is not utilized early in life. As lung function deteriorates,CF patients can develop pulmonary hypertension, chronic bronchitis, andchronic dilation of the bronchioles (bronchiectasis). Lung abscess arevery common. Death usually occurs from severe infection, pneumonia, orheart failure.

Cystic fibrosis is exclusively heritable as both parents must carry therecessive genes for a child to acquire the disease. At the geneticlevel, cystic fibrosis is most often the result of an in-frame deletionof three base pairs in the DNA. Cystic fibrosis results from theproduction of an abnormal form of a protein called cystic fibrosistransmembrane conductance regulator (CFTR). CFTR functions intransporting chloride ions across epithelial cells found in the lung andintestinal tract. In CF patients, CFTR does not function properly,causing accumulation of ions inside epithelial cells. Since waterfollows ions by osmosis, this results in water depletion and viscousmucus on the surface of alveoli. The most common CFTR proteinabnormality is a mutation termed ΔF508, which is characterized by the3-bp deletion of the DNA basepair sequence at chromosome location7q31.1-31.2 that codes for the amino acid, phenylalanine.

In addition to pulmonary infections, most people with CF also haveproblems with digestion, particularly the digestion of fats. This leadsto malabsorption and difficulty gaining and maintaining weight, which inturn affects overall health. This is due to the abnormally sticky mucusthat blocks the release of digestive enzymes from the pancreas.Pancreatic insufficiency is treated with supplemental enzymes. Usuallywater-miscible forms of the fat-soluble vitamins A, D, E, and K arerequired as the decreased fat absorption can lead to deficiencies ofthese vitamins.

CF patients also have an increased incidence of diabetes mellitusbecause of the pancreatic blockage. The chronic blocking causes theIslets of Langerhans to degrade over time and decrease insulinproduction, causing hyperglycemia. There is also evidence that patientswith CF become more resistant to the insulin that is produced, this canbe triggered by infections or treatment with corticosteroids. Diabetesin CF patients is commonly referred to as CFRD, cystic fibrosis relateddiabetes. A typical diabetic diet is not feasible and therefore insulindoses are instead adjusted to fit the typical high-calorie/high-fat CFdiet.

Many CF patients, to some degree, experience the widening of the tips oftheir fingers, known as “clubbing”. The condition affects fingers andtoes, and results in the tip of the digit being round and enlarged. Thiscan also be seen in people with COPD or severe heart disease. Sincepeople with CF are prone to poor absorption of nutrients, osteoporosiscan occur in early adulthood due to low bone density. It is importantfor people with CF to have regular dual energy X-ray absorptiometry(DEXA) scans to measure bone density and begin treatment if needed. Whendiagnosed early, treatment can help prevent more serious complications.

Some CF patients have hearing loss as a side effect of long-term use ofthe -mycin/-micin group of drugs, such as Tobramycin, which is used tocombat lung infections. Although this side-effect is well-known andunderstood, these particular antibiotics are of high value in thetreatment of CF patients, and often the hearing loss must be considereda necessary trade-off in order to preserve life and health. CF occursprimarily in individuals of central and western European origin. In theUnited States, the median age at death has increased from 8.4 years ofage in 1969 to 14.3 years of age in 1998. The mean age of death hasincreased from 14 years in 1969 to 32.4 years of age in 2003 (CysticFibrosis Foundation). A major contributor to the significant increase inlife expectancy is improved antibiotic treatment of chronic respiratorytract infections in CF subjects (Goss and Rosenfeld 2004) as well asimproved nutrition and earlier diagnosis.

A major factor in the respiratory health of CF subjects is acquisitionof chronic Pseudomonas aeruginosa infections. The infection rate with P.aeruginosa increases with age and by age 18 years, 80% of CF subjects inthe U.S. are infected. The difficulties treating this infection aremultifactorial, including poor penetration of antibiotics into sites ofinfection including mucus plugs, inactivation of antibiotics by CFsputum, growth of bacteria in a biofilm, changes in phenotype includingconversion to a mucoid form of P. aeruginosa, and emergence ofmulti-drug resistance (Chmiel and Davis 2003; Gibson, Burns et al.2003). The cornerstone of pulmonary therapy is optimizing treatment ofP. aeruginosa as infection with this pathogen is associated with a poorclinical outcome (Doring, Conway et al. 2000; Chmiel and Davis 2003;Gibson, Burns et al. 2003; Gibson, Emerson et al. 2003).

One of the current approaches to management of chronic P. aeruginosainfection in humans with CF includes the use of suppressive therapy withinhaled tobramycin (TOBI®) Inhaled tobramycin, 300 mg, administeredtwice a day for cycles of 28 days followed by 28 days off drug has beenshown to reduce P. aeruginosa colony counts, increase FEV₁% predicted,reduce hospitalizations, and decrease antibiotic use (Ramsey, Pepe etal. 1999). Nevertheless, patients have to be dosed twice a day forapproximately 15-20 minute inhalation periods per dose.

Daily chest physiotherapy and aerosol breathing treatments are verycommonly prescribed for CF patients. Typical physical therapy involvesmanual chest percussion (pounding), positive pressure techniquesand/devices or possibly using a device such as the ThAIRapy Vest or theIntrapulmonary Percussive Ventilator (IPV) to achieve the same effect:loosening of the thick mucus. Aerosolized medicines commonly giveninclude albuterol, ipratropium bromide and Pulmozyme to loosensecretions and decrease inflammation. It was found that CFers who surfwere healthier; consequently, some hospitals use a nebulized 6%-10%Saline solution on those CFers who do not have asthma to loosen thesecretions. Inhaled aminoglycoside antibiotics are sometimes given tofight infections. A number of pharmacological agents that help mucosalclearance are being used. N-acetylcysteine that solubilizes mucusglycoprotein, however, has not proved to be significantly effective.Recombinant human DNAse decreases the viscosity of sputum by degradingthe concentrated amount of DNA in the sputum of CF patients. DNAsetreatment has been beneficial in increasing airflow during short-termuse, and has also prolonged the interval between episodes of pulmonaryexacerbations.

CF patients are typically hospitalized somewhat regularly, often every 6months depending on the severity of the case. Patients often haveintravenous antibiotics through a PICC line, Central Line, orPort-a-Caths.

Cystic fibrosis can also lead to bronchiectasis. Bronchiectasis is anabnormal stretching and enlarging of the respiratory passages caused bymucus blockage. When the body is unable to get rid of mucus, mucusbecomes stuck and accumulates in the airways. The blockage andaccompanying infection cause inflammation, leading to the weakening andwidening of the passages. The weakened passages can become scarred anddeformed, allowing more mucus and bacteria to accumulate, resulting in acycle of infection and blocked airways. Bronchiectasis is a disease thatcauses localized, irreversible dilatation of part of the bronchial tree.Involved bronchi are dilated, inflamed, and easily collapsible,resulting in airflow obstruction and impaired clearance of secretions.Bronchiectasis is associated with a wide range of disorders, but itusually results from necrotizing bacterial infections, such asinfections caused by the Staphylococcus or Klebsiella species orBordatella pertussis.

Bronchiectasis is one of the chronic obstructive pulmonary diseases(COPD) and it can be complicated by emphysema and bronchitis. Thedisease is commonly misdiagnosed as asthma or pneumonia. Bronchiectasiscan develop at any age, begins most often in childhood, but symptoms maynot be apparent until much later. Bronchiectasis can occur as part of abirth defect, such as primary ciliary dyskinesia or cystic fibrosis.About 50% of all cases of bronchiectasis in the U.S. result from cysticfibrosis. It can also develop after birth as a result of injury or otherdiseases, like tuberculosis, pneumonia and influenza.

Dilation of the bronchial walls results in airflow obstruction andimpaired clearance of secretions because the dilated areas interruptnormal air pressure of the bronchial tubes, causing sputum to poolinside the dilated areas instead of being pushed upward. The pooledsputum provides an environment conducive to the growth of infectiouspathogens, and these areas of the lungs are thus very vulnerable toinfection. The more infections that the lungs experience, the moredamaged the lung tissue and alveoli become. When this happens, thebronchial tubes become more inelastic and dilated, which creates aperpetual, destructive cycle within this disease.

There are three types of bronchiectasis, varying by level of severity.Fusiform (cylindrical) bronchiectasis (the most common type) refers tomildly inflamed bronchi that fail to taper distally. In varicosebronchiectasis, the bronchial walls appear beaded, because areas ofdilation are mixed with areas of constriction. Saccular (cystic)bronchiectasis is characterized by severe, irreversible ballooning ofthe bronchi peripherally, with or without air-fluid levels. Chronicproductive cough is prominent, occurring in up to 90% of patients withbronchiectasis. Sputum is produced on a daily basis in 76% of patients.

In addition to CF, other genetic causes or contributing factors tobronchiectasisis include Kartagener syndrome, Young's syndrome, alpha1-antitrypsin deficiency, and Primary immunodeficiencies. Acquiredbronchiectasis occurs more frequently, with one of the biggest causesbeing tuberculosis. A especially common cause of the disease in childrenis Acquired Immunodeficiency Syndrome, stemming from the humanimmunodeficiency virus. Other causes of bronchiectasis includerespiratory infections, obstructions, inhalation and aspiration ofammonia, and other toxic gases, pulmonary aspiration, alcoholism, heroinuse and allergies. Cigarette smoking may also contribute tobronchiectasis.

The diagnosis of bronchiectasis is based on the review of clinicalhistory and characteristic patterns in high-resolution CT scan findings.Such patterns include “tree-in-bud” abnormalities and cysts withdefinable borders. Bronchiectasis may also be diagnosed without CT scanconfirmation if clinical history clearly demonstrates frequent,respiratory infections, as well confirmation of an underlying problemvia blood work and sputum culture samples.

Symptoms include coughing (worsened when lying down), shortness ofbreath, abnormal chest sounds, weakness, weight loss, and fatigue. Withinfections the mucus may be discolored, foul smelling and may containblood. Symptom severity varies widely from patient to patient andoccasionally, a patient is asymptomatic.

Treatment of bronchiectasis is aimed at controlling infections andbronchial secretions, relieving airway obstruction, and preventingcomplications. This includes prolonged usage of antibiotics to preventdetrimental infections, as well as eliminating accumulated fluid withpostural drainage and chest physiotherapy. Surgery may also be used totreat localized bronchiectasis, removing obstructions that could causeprogression of the disease.

Inhaled steroid therapy that is consistently adhered to can reducesputum production and decrease airway constriction over a period of timewill prevent progression of bronchiectasis. One commonly used therapy isbeclometasone dipropionate, also used in asthma treatment. Use ofinhalers such as Albuterol (Salbutamol), Fluticasone (Flovent/Flixotide)and Ipratropium (Atrovent) may help reduce likelihood of infection byclearing the airways and decreasing inflammation.

Mannitol dry inhalation powder, under the name Bronchitol, has beenapproved by the FDA for use in Cystic Fibrosis patients withBronchiectasis. The original orphan drug indication approved in February2005 allowed its use for the treatment of bronchiectasis. The originalapproval was based on the results of phase 2 clinical studies showingthe product to be safe, well-tolerated, and effective for stimulatingmucus hydration/clearance, thereby improving quality of life in patientswith chronic obstructive lung diseases like Bronchiectasis. Long-termstudies are underway as of 2007 to ensure the safety and effectivenessof the treatment.

Bronchiectasis patients are often given antibiotics for infection andbronchodilator medicines to open passages. Sometimes antibiotics areprescribed for a long period to prevent recurring infections, especiallyin people who have cystic fibrosis. There are also physical therapytechniques to help clear mucus. Lung transplants are also an option forsevere cases. Fatalities are uncommon but may result from massivehemorrhage. If lung infections are treated immediately, bronchiectasisis less likely to develop.

Pneumonia is an illness of the lungs and respiratory system in which thealveoli (microscopic air-filled sacs of the lung responsible forabsorbing oxygen from the atmosphere) become inflamed and flooded withfluid. Pneumonia can result from a variety of causes, includinginfection with bacteria, viruses, fungi, or parasites, and chemical orphysical injury to the lungs. Typical symptoms associated with pneumoniainclude cough, chest pain, fever, and difficulty in breathing.Diagnostic tools include x-rays and examination of the sputum.

Therefore, there is a need for therapies to treat pulmonary disorders,including CF, pulmonary infections, COPD, bronchiectasis and others.Additionally, there is a need to improve lung function in patientshaving such disorders.

SUMMARY OF THE INVENTION

The present invention relates in part to a method of treating apulmonary disorder in a patient comprising administering to the patientan effective dose of a nebulized liposomal amikacin formulation for atleast one treatment cycle, wherein:

-   -   the treatment cycle comprises an administration period of 15 to        75 days, followed by an off period of 15 to 75 days;    -   and the effective dose comprises 100 to 2500 mg of amikacin        daily during the administration period.

In some embodiments, the treatment cycle is administered to the patientat least twice. In some embodiments, the administration period is 15 to35 days, or 20 to 35 days. In other embodiments, the administrationperiod is about 28 days. In some embodiments, the off period is 15 to 35days, or 20 to 35 days. In other embodiments, the off period is about 28days. In still other embodiments, the off period is of 25 to 75 days, 35to 75 days, or 45 to 75 days. In other embodiments, the off period isabout 56 days.

In some embodiments, the administration period is about 28 days and theoff period is about 28 days, while in other embodiments, theadministration period is about 28 days and the off period is about 56days.

In some embodiments, the effective dose comprises 250 to 1,500 mg ofamikacin, 250 to 1000 mg of amikacin, or about 280 to about 560 mg ofamikacin. In other embodiments, the effective dose is about 280 or about560 mg of amikacin.

In some embodiments, the pulmonary disorder is selected from the groupconsisting of chronic obstructive pulmonary disease, bronchiectasis,pulmonary infection, cystic fibrosis, alpha-1-antitrypsin enzymedeficiency and a combination thereof. In other embodiments, thepulmonary condition is a bacterial pulmonary infection, such as a P.aeruginosa infection. In some embodiments, the pulmonary condition isbronchiectasis.

In some embodiments, the patient has a serum C_(max) of amikacin of lessthan about 10 mcg/mL during the administration period. In otherembodiments, the patient has a sputum C_(max) of amikacin of at least1000 mcg per gram of sputum either during the administration, for atleast 15 days after the administration.

In some embodiments, the patient has a reduction in log₁₀ CFU of thebacterial infection in the lungs of at least 0.5 for at least 15 daysafter the administration period ends. In other embodiments, thereduction in the log₁₀ CFU is at least 1.0.

In some embodiments, the patient experiences an improvement in lungfunction for at least 15 days after the administration period ends. Forexample, the patient may experience an increase in FEV₁, an increase inblood oxygen saturation, or both. In some embodiments, the patient hasan FEV₁ that is increased by at least 5% over the FEV₁ prior to thetreatment cycle. In other embodiments, FEV₁ is increased by 5 to 50%. Inother embodiments, FEV₁ is increased by 25 to 500 mL over FEV₁ prior tothe treatment cycle. In some embodiments, blood oxygen saturation isincreased by at least 1% over oxygen saturation prior to the treatmentcycle.

In some embodiments, the length of time to a pulmonary exacerbation isat least 20 days from the last day of administration. In otherembodiments, the length of time to a rescue treatment is at least 25days from the last day of the administration.

In some embodiments, the liposomal amikacin formulation comprises alipid selected from the group consisting of egg phosphatidylcholine(EPC), egg phosphatidylglycerol (EPG), egg phosphatidylinositol (EPI),egg phosphatidylserine (EPS), phosphatidylethanolamine (EPE),phosphatidic acid (EPA), soy phosphatidylcholine (SPC), soyphosphatidylglycerol (SPG), soy phosphatidylserine (SPS), soyphosphatidylinositol (SPI), soy phosphatidylethanolamine (SPE), soyphosphatidic acid (SPA), hydrogenated egg phosphatidylcholine (HEPC),hydrogenated egg phosphatidylglycerol (HEPG), hydrogenated eggphosphatidylinositol (HEPI), hydrogenated egg phosphatidylserine (HEPS),hydrogenated phosphatidylethanolamine (HEPE), hydrogenated phosphatidicacid (HEPA), hydrogenated soy phosphatidylcholine (HSPC), hydrogenatedsoy phosphatidylglycerol (HSPG), hydrogenated soy phosphatidylserine(HSPS), hydrogenated soy phosphatidylinositol (HSPI), hydrogenated soyphosphatidylethanolamine (HSPE), hydrogenated soy phosphatidic acid(HSPA), dipalmitoylphosphatidylcholine (DPPC),dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol(DMPG), dipalmitoylphosphatidylglycerol (DPPG),distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol(DSPG), dioleylphosphatidyl-ethanolamine (DOPE),palmitoylstearoylphosphatidyl-choline (PSPC),palmitoylstearolphosphatidylglycerol (PSPG),mono-oleoyl-phosphatidylethanolamine (MOPE), cholesterol, ergosterol,lanosterol, tocopherol, ammonium salts of fatty acids, ammonium salts ofphospholipids, ammonium salts of glycerides, myristylamine,palmitylamine, laurylamine, stearylamine, dilauroyl ethylphosphocholine(DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoylethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP),N-(2,3-di-(9-(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammoniumchloride (DOTMA), 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane(DOTAP), phosphatidyl-glycerols (PGs), phosphatidic acids (PAs),phosphatidylinositols (PIs), phosphatidyl serines (PSs),distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylacid(DMPA), dipalmitoylphosphatidylacid (DPPA), distearoylphosphatidylacid(DSPA), dimyristoylphosphatidylinositol (DMPI),dipalmitoylphosphatidylinositol (DPPI), distearoylphospatidylinositol(DSPI), dimyristoylphosphatidylserine (DMPS),dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine(DSPS), and mixtures thereof. In other embodiments, the liposomalamikacin formulation comprises a phospholipid and a sterol, such as DPPCand cholesterol. In other embodiments, the liposomal amikacinformulation comprises DPPC and cholesterol in about a 2 to 1 ratio byweight. In some embodiments, the liposomal amikacin formulation has alipid to drug ratio of about 0.5 to about 1.0, about 0.5 to 0.7, orabout 0.6 by weight.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts mass distribution of Liposomal Amikacin nebulizatecollected on impactor stages as a function of cutoff diameter. The threeLiposomal Amikacin lots of Table 15 legend (designated as 1, 2, and 3)were used with the eFlow nebulizer and ACI system (solid symbols) or theLC Star nebulizer and NGI system (open symbols).

FIG. 2 depicts reduction in the Log₁₀ CFU/Lungs of Rats after Inhalationof Liposomal Amikacin 75 mg/mL or Tobramycin. The symbols represent theLog₁₀ CFU/lungs of each rat 18 days after the instillation of PA3064 inagar beads and 3 days after the last inhalation session of saline or oneof the above antibiotics. The values at 2.0 Log₁₀ CFU represent thelower limit of detection of bacteria in the lung in the method. The barrepresents the mean of each group. The means and standard deviations andtwo-tail t-test results were calculated using Excel software byMicrosoft.

FIG. 3 depicts reduction in the Log₁₀ CFU/lungs of rats after Inhalationof Liposomal Amikacin and Tobramycin for 28 days. Equivalent doses ofthe above antibiotics were given by inhalation therapy but on differentschedules. Tobramycin was given BID daily for a total of 104 min per dayfor 28 days. Liposomal Amikacin was given once daily for 80 min for 28days (Q1D×28) as was saline. Liposomal Amikacin was also given oncedaily for 160 min every other day for 28 days (Q2D×14) or once daily for160 min for 14 consecutive days (Q1D×14) then just observed until therats were euthanized. The symbols represent the Log₁₀ CFU/lungs of eachrat 35 days after the instillation of P. aeruginosa 3064 in agar beads.The means and standard deviations and two-tail t-test were calculatedusing Excel software by Microsoft).

FIG. 4 depicts the study designs for Study 4, wherein patients receivedliposomal amikacin daily for 28 days, followed by monitoring for a 28day period after the last day of administration.

FIG. 5 depicts a graph showing the percent increase in oxygen saturationover baseline in pediatric patients receiving a 280 mg dose of amikacincompared to a placebo.

FIG. 6 depicts a graph showing the oxygen saturation in pediatricpatients receiving a 560 mg dose of amikacin compared to a placebo.

FIG. 7a depicts a graph of lung function change by age as measured byFEV1 in the placebo group. Data for placebo for both 280 and 560 mgamikacin arms of the study were pooled and divided by age. Also, datafor Arikace™ for 280 and 560 mg amikacin arms were pooled and divided byage.

FIG. 7b depicts the lung function change by age in the patientsreceiving inhaled liposomal amikacin.

FIG. 8 depicts a graph comparing the change in FEV1 (measured in mL) inthe 560 mg and 280 mg amikacin groups, and the placebo group.

FIG. 9 depicts a graph of the change in FEV1 as a percent relative tobaseline in the 560 mg amikacin, 280 mg amikacin, and placebo groups.

FIG. 10 depicts a graph of the Log CFU change in all patients.

FIG. 11 depicts a graph of the Log CFU change for mucoid strains.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

For convenience, before further description of the present invention,certain terms employed in the specification, examples and appendedclaims are collected here. These definitions should be read in light ofthe remainder of the disclosure and understood as by a person of skillin the art. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by a person ofordinary skill in the art.

The term “pulmonary disorder” refers to any disease, ailment, or otherunhealthy condition related to the respiratory tract of a subject,particularly the lungs of a subject. Generally pulmonary distressresults in difficulty of breathing.

The term “treating” is art-recognized and refers to curing as well asameliorating at least one symptom of any condition or disease.

The term “prophylactic” or “therapeutic” treatment is art-recognized andrefers to administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, i.e., it protects thehost against developing the unwanted condition, whereas if administeredafter manifestation of the unwanted condition, the treatment istherapeutic (i.e., it is intended to diminish, ameliorate or maintainthe existing unwanted condition or side effects therefrom).

The terms “therapeutically effective dose” and “therapeuticallyeffective amount” refer to that amount of a compound that results inprevention or amelioration of symptoms in a patient or a desiredbiological outcome, e.g., improved clinical signs, delayed onset ofdisease, reduced levels of bacteria, etc.

The term “FEV₁” is well known in the art as a measure of lung function,and refers to the forced expiratory volume in one second. The FEV₁values used herein are measured in mL's, and also in terms of percentchange from baseline, e.g., a change from pre-treatment values.

A “patient,” “subject” or “host” to be treated by the subject method maymean either a human or non-human animal.

The term “mammal” is known in the art, and exemplary mammals includehumans, primates, bovines, porcines, canines, felines, and rodents(e.g., mice and rats).

The term “bioavailable” is art-recognized and refers to a form of thesubject invention that allows for it, or a portion of the amountadministered, to be absorbed by, incorporated to, or otherwisephysiologically available to a subject or patient to whom it isadministered.

The term “pharmaceutically-acceptable salts” is art-recognized andrefers to the relatively non-toxic, inorganic and organic acid additionsalts of compounds, including, for example, those contained incompositions of the present invention.

The term “pharmaceutically acceptable carrier” is art-recognized andrefers to a pharmaceutically-acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material, involved in carrying or transporting anysubject composition or component thereof from one organ, or portion ofthe body, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the subjectcomposition and its components and not injurious to the patient. Someexamples of materials which may serve as pharmaceutically acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations.

II. Liposomal Amikacin

Liposomal amikacin formulations useful in the presently disclosedmethods can be prepared as described, for example, in U.S. PublicationNo. 20060073198 or 20080089927, both of which are hereby incorporated byreference. Generally, amikacin is used in the form of a pharmaceuticallyacceptable salt, for example the sulfate salt of amikacin.

The lipids used in the compositions of the present invention can besynthetic, semi-synthetic or naturally-occurring lipids, includingphospholipids, tocopherols, steroids, fatty acids, glycoproteins such asalbumin, anionic lipids and cationic lipids. The lipids may be anionic,cationic, or neutral. In one embodiment, the lipid formulation issubstantially free of anionic lipids, substantially free of cationiclipids, or both. In one embodiment, the lipid formulation comprises onlyneutral lipids. In another embodiment, the lipid formulation is free ofanionic lipids or cationic lipids or both. In another embodiment, thelipid is a phospholipid. Phospholipids include egg phosphatidylcholine(EPC), egg phosphatidylglycerol (EPG), egg phosphatidylinositol (EPI),egg phosphatidylserine (EPS), phosphatidylethanolamine (EPE), and eggphosphatidic acid (EPA); the soya counterparts, soy phosphatidylcholine(SPC); SPG, SPS, SPI, SPE, and SPA; the hydrogenated egg and soyacounterparts (e.g., HEPC, HSPC), other phospholipids made up of esterlinkages of fatty acids in the 2 and 3 of glycerol positions containingchains of 12 to 26 carbon atoms and different head groups in the 1position of glycerol that include choline, glycerol, inositol, serine,ethanolamine, as well as the corresponding phosphatidic acids. Thechains on these fatty acids can be saturated or unsaturated, and thephospholipid can be made up of fatty acids of different chain lengthsand different degrees of unsaturation. In particular, the compositionsof the formulations can include dipalmitoylphosphatidylcholine (DPPC), amajor constituent of naturally-occurring lung surfactant as well asdioleoylphosphatidylcholine (DOPC). Other examples includedimyristoylphosphatidylcholine (DMPC) anddimyristoylphosphatidylglycerol (DMPG) dipalmitoylphosphatidcholine(DPPC) and dipalmitoylphosphatidylglycerol (DPPG)distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol(DSPG), dioleylphosphatidylethanolamine (DOPE) and mixed phospholipidslike palmitoylstearoylphosphatidylcholine (PSPC) andpalmitoylstearoylphosphatidylglycerol (PSPG), driacylglycerol,diacylglycerol, seranide, sphingosine, sphingomyelin and single acylatedphospholipids like mono-oleoyl-phosphatidylethanol amine (MOPE).

The lipids used can include ammonium salts of fatty acids, phospholipidsand glycerides, phosphatidylglycerols (PGs), phosphatidic acids (PAs),phosphotidylcholines (PCs), phosphatidylinositols (PIs) and thephosphatidylserines (PSs). The fatty acids include fatty acids of carbonchain lengths of 12 to 26 carbon atoms that are either saturated orunsaturated. Some specific examples include: myristylamine,palmitylamine, laurylamine and stearylamine, dilauroylethylphosphocholine (DLEP), dimyristoyl ethylphosphocholine (DMEP),dipalmitoyl ethylphosphocholine (DPEP) and distearoylethylphosphocholine (DSEP), N-(2,3-di-(9(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride (DOTMA)and 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP). Examples ofPGs, PAs, PIs, PCs and PSs include DMPG, DPPG, DSPG, DMPA, DPPA, DSPA,DMPI, DPPI, DSPI, DMPS, DPPS and DSPS, DSPC, DPPG, DMPC, DOPC, egg PC.

In another embodiment, the liposome comprises a lipid selected from thegroup consisting of phosphatidylcholines (PCs), phosphatidyl-glycerols(PGs), phosphatidic acids (PAs), phosphatidylinositols (PIs), andphosphatidyl serines (PSs).

In another embodiment, the lipid is selected from the group consistingof: egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), eggphosphatidylinositol (EPI), egg phosphatidylserine (EPS),phosphatidylethanolamine (EPE), phosphatidic acid (EPA), soyphosphatidylcholine (SPC), soy phosphatidylglycerol (SPG), soyphosphatidylserine (SPS), soy phosphatidylinositol (SPI), soyphosphatidylethanolamine (SPE), soy phosphatidic acid (SPA),hydrogenated egg phosphatidylcholine (HEPC), hydrogenated eggphosphatidylglycerol (HEPG), hydrogenated egg phosphatidylinositol(HEPI), hydrogenated egg phosphatidylserine (HEPS), hydrogenatedphosphatidylethanolamine (HEPE), hydrogenated phosphatidic acid (HEPA),hydrogenated soy phosphatidylcholine (HSPC), hydrogenated soyphosphatidylglycerol (HSPG), hydrogenated soy phosphatidylserine (HSPS),hydrogenated soy phosphatidylinositol (HSPI), hydrogenated soyphosphatidylethanolamine (HSPE), hydrogenated soy phosphatidic acid(HSPA), dipalmitoylphosphatidylcholine (DPPC),dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol(DMPG), dipalmitoylphosphatidylglycerol (DPPG),distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol(DSPG), dioleylphosphatidyl-ethanolamine (DOPE),palmitoylstearoylphosphatidyl-choline (PSPC),palmitoylstearolphosphatidylglycerol (PSPG),mono-oleoyl-phosphatidylethanolamine (MOPE), tocopherol, ammonium saltsof fatty acids, ammonium salts of phospholipids, ammonium salts ofglycerides, myristylamine, palmitylamine, laurylamine, stearylamine,dilauroyl ethylphosphocholine (DLEP), dimyristoyl ethylphosphocholine(DMEP), dipalmitoyl ethylphosphocholine (DPEP) and distearoylethylphosphocholine (DSEP),N-(2,3-di-(9-(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammoniumchloride (DOTMA), 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane(DOTAP), distearoylphosphatidylglycerol (DSPG),dimyristoylphosphatidylacid (DMPA), dipalmitoylphosphatidylacid (DPPA),distearoylphosphatidylacid (DSPA), dimyristoylphosphatidylinositol(DMPI), dipalmitoylphosphatidylinositol (DPPI),distearoylphospatidylinositol (DSPI), dimyristoylphosphatidylserine(DMPS), dipalmitoylphosphatidylserine (DPPS),distearoylphosphatidylserine (DSPS), and mixtures thereof.

In another embodiment, the liposome comprises a phosphatidylcholine. Thephosphatidylcholine may be unsaturated, such as DOPC or POPC, orsaturated, such as DPPC. In another embodiment, the liposome does notinclude a sterol. In one embodiment, the liposome consists essentiallyof a phosphatidylcholine and a sterol. In another embodiment, theliposome consists essentially of DPPC and cholesterol.

Liposomes or lipid antiinfective formulations composed ofphosphatidylcholines, such as DPPC, aid in the uptake by the cells inthe lung such as the alveolar macrophages and helps to sustain releaseof the antiinfective agent in the lung (Gonzales-Rothi et al. (1991)).The negatively charged lipids such as the PGs, PAs, PSs and PIs, inaddition to reducing particle aggregation, can play a role in thesustained release characteristics of the inhalation formulation as wellas in the transport of the formulation across the lung (transcytosis)for systemic uptake.

While not being bound by any particular theory, it is believed that whenthe lipid comprises a neutral lipid, and does not comprise a negativelycharged or positively charged phospholipid, the liposomal formulationhas improved uptake by the lungs. For example, the liposome my haveimproved penetration into a biofilm or mucus layer when the lipidcomprises only neutral lipids. Exemplary neutral lipids include theaforementioned phosphatidylcholines, such as DPPC and sterols, such ascholesterol.

IV. Methods of Treatment

The present invention is directed to methods of treating a pulmonarycondition in a subject need thereof comprising administering to thesubject and effective amount of any one of the aforementioned liposomalantibiotic formulations. In some embodiments, the pulmonary condition isa bacterial infection. In some embodiments, the method comprisesadministering to a patient in need thereof an effective amount of aliposomal amikacin formulation (also referred to herein as “liposomalamikacin”) by inhalation daily. In some embodiments, the administrationby inhalation comprises nebulizing the liposomal formulation.

In some embodiments, the liposomal amikacin formulation is administereddaily for a period of time, followed by second period of time (an “off”period) wherein no liposomal formulation is administered. For example,in some embodiments, the method of treating a pulmonary disordercomprises administering to the patient an effective dose of a nebulizedliposomal amikacin formulation for at least one treatment cycle,wherein:

-   -   the treatment cycle comprises an administration period of 15 to        75 days, followed by an off period of 15 to 75 days;    -   and the effective dose comprises 100 to 2500 mg of amikacin        daily during the administration period.        In some embodiments, the aforementioned treatment cycle is        administered to the patient at least twice. In other        embodiments, the treatment cycle may be administered 3, 4, 5, 6,        or more times.

During the administration period, liposomal amikacin is administereddaily. In some embodiments, liposomal amikacin can be administered everyother day or every third day during the administration period. Asexplained above, the administration period can be 15 to 75 days. In someembodiments, the administration period is 15 to 35 days, or 20 to 35days. In other embodiments, the administration period is 20 to 30 days,25 to 35 days or 25 to 30 days. In other embodiments, the administrationperiod is about 25, 26, 27, 28, 29 or 30 days. In another embodiment,the administration period is about 28 days.

During the off period the liposomal amikacin formulation is notadministered to the patient. In some embodiments, the off period is 15days or longer, for example, 15 to 75 days, 15 to 35 days, or 20 to 35days. In other embodiments, the off period is 20 to 30 days, 25 to 35days or 25 to 30 days. In other embodiments, the off period is about 25,26, 27, 28, 29 or 30 days. In other embodiments, the off period is about28 days, while in still other embodiments, the off period is at least 29days.

In some embodiments, the off period is of 25 to 75 days, 35 to 75 days,or 45 to 75 days. In other embodiments, the off period is 50 to 75 days,50 to 70 days, 50 to 65 days or 50 to 60 days. In other embodiments, theoff period is about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 days,while in other embodiments, the off period is about 56 days.

In some embodiments, the administration period is about 28 days and theoff period is about 28 days, while in other embodiments, theadministration period is about 28 days and the off period is about 56days.

In some embodiments, the effective dose comprises 250 to 1,500 mg ofamikacin, 250 to 1000 mg of amikacin, 250 to 750 mg of amikacin, or 250to 700 mg amikacin each day of the administration period. In otherembodiments, the effective dose is about 280 to about 560 mg ofamikacin. In other embodiments, the effective dose is about 230 mg toabout 330 mg, or about 510 mg to about 610 mg. In other embodiments, theeffective dose of amikacin is about 100, 150, 200, 250, 300, 250, 400,450, 500, 550, 600, 650, 700 or 750 mg of amikacin daily. In otherembodiments, the effective dose is about 280 or about 560 mg ofamikacin.

In some embodiments, the administration period is about 28 days, and thedose is about 280 to about 560 mg of amikacin. In other embodiments, theadministration period is about 28 days, the off period is about 28 days,and the dose is about 280 to about 560 mg. In other embodiments, theadministration period is about 28 days, the off period is about 56 days,and the dose is about 280 o about 560 mg.

In some embodiments, the pulmonary disorder is selected from the groupconsisting of chronic obstructive pulmonary disease, bronchiectasis,pulmonary infection, cystic fibrosis, alpha-1-antitrypsin enzymedeficiency and a combination thereof. In some embodiments, the pulmonarycondition is cystic fibrosis. In other embodiments, the pulmonarycondition is a bacterial pulmonary infection, Pseudomonas (e.g., P.aeruginosa, P. paucimobilis, P. putida, P. fluorescens, and P.acidovorans), staphylococcal, Methicillin-resistant Staphylococcusaureus (MRSA), streptococcal (including by Streptococcus pneumoniae),Escherichia coli, Klebsiella, Enterobacter, Serratia, Haemophilus,Yersinia pesos, Burkholderia pseudomallei, B. cepacia, B. gladioli, B.multivorans, B. vietnamiensis, Mycobacterium tuberculosis, M. aviumcomplex (MAC)(M. avium and M. intracellulare), M. kansasii, M. xenopi,M. marinum, M. ulcerans, or M. fortuitum complex (M. fortuitum and M.chelonei) infections. In some embodiments, the infection is a P.aeruginosa infection, while in other embodiments, the infection is anon-tuberculous mycobacterial infection. The pulmonary infection may ormay not be associated with cystic fibrosis. Thus, in some embodiments,the pulmonary condition is both cystic fibrosis and a pulmonaryinfections such as P. aeruginosa. In other embodiments, the pulmonaryconditions is bronchiectasis. The bronchiectasis may or may not beassociated with cystic fibrosis.

The present method provides advantageous levels of amikacin at the siteof the pulmonary disorder, while limiting systemic exposure to the drug,and also provides a sustained benefit to the subject for surprisinglyextended periods of time. While not being bound by any particulartheory, it is believed that administration of liposomal amikacin inaccordance the with methods described herein results a “depot” effect inthe lungs of the subject. Specifically, it is believed that the liposomeparticles are small enough and contain an appropriate lipid formulationto penetrate and diffuse through CF sputum and into the bacterialbiofilm. The liposomes shield the entrapped cationic amikacin in neutralliposomes to minimize electrostatic interaction with the negativelycharged sputum/biofilm, which would otherwise reduce itsbioavailability. In addition, there are P. aeruginosa derived virulencefactors (rhamnolipids) (Davey et al. 2003), which release amikacin theliposomes. Therefore, it is hypothesized that relatively highconcentrations of drug can be delivered locally to the bacterialmacro-colony environment.

Additionally, it is believed that inhalation of liposomal amikacin leadsto a dose dependent recruitment of macrophages as an adaptive responseto inhalation of drug/lipid formulation. The presence of alveolarmacrophages (which have been shown to be functionally normal inliposomal amikacin treated rats) may be particularly beneficial in CFpatients. CF patients are known to have reduced number of macrophages intheir lungs and possibly with poor functionality, which may contributeto the chronicity of P. aeruginosa lung infection, and to the higherprevalence of non-tuberculous mycobacterial infection in thispopulation. The dose dependent recruitment of macrophages may alsocontribute to the sustained effects observed using the methods of thepresent invention. Specifically, the macrophages in the lung may take upliposomal amikacin, and then remain in the lung for a period of time,followed by release of the liposomal amikacin by the macrophages. Aclinical study (described in the exemplification below) of liposomalamikacin in CF patients chronically infected with P. aeruginosa hasdemonstrated safety, tolerability and dose dependent improvement in lungfunction and respiratory symptoms; and reduction of sputum bacterialdensity at the end of 28 days of treatment. This improvement in lungfunction was sustained for at least 28 days after completion oftreatment (Day 56) with a 560 mg dose of liposomal amikacin, indicatinga sustained treatment effect.

The present method thus provides, in some embodiments, advantageouslevels of amikacin in the blood and in the sputum. For example, themethods provides relatively low systemic exposure to amikacin, whileproviding high, sustained levels of amikacin at the site of thepulmonary condition. For example, in some embodiments, the patient has aserum C_(max) of amikacin of less than about 25 mcg/mL during theadministration period. In other embodiments, the serum C_(max) is lessthan 20, 15, 10, 5 or 2 mcg/mL during the administration period.

In some embodiments, the patient has a sputum C_(max) of amikacin of atleast about 500 mcg per gram of sputum either during the administration,or for a sustained period of time, such as at least 15 days, after theadministration. In other embodiments, the sputum C_(max) of amikacin isat least 750, 1000, 1500, 2000, 2500, 3000 or 3500 mcg per gram ofsputum.

When the pulmonary disorder includes a pulmonary infection, the presentinvention also provides a reduction in the colony forming units of thebacteria in the lung for a sustained period of time. For example, theCFU's are reduced compared to a baseline value. In some embodiments, thepatient has a reduction in log₁₀ CFU of the bacterial infection in thelungs of at least about 0.5 for at least 15 days after theadministration period ends. In other embodiments, the reduction in thelog₁₀ CFU is at least by 1.0, 1.5, 2.0 or 2.5. Pseudomonas infections,in particular, can form large colonies, known as “mucoid” Pseudomonas,particularly in patients with cystic fibrosis. In some embodiments, theCFU's are reduced as described above in a mucoid strain of a Pseudomonasinfection.

In some embodiments, the patient experiences an improvement in lungfunction for at least 15 days after the administration period ends. Forexample, the patient may experience an increase in the forced expiratoryvolume in one second (FEV₁), an increase in blood oxygen saturation, orboth. In some embodiments, the patient has an FEV₁ that is increased byat least 5% or at least 10% over the FEV₁ prior to the treatment cycle.In other embodiments, FEV₁ is increased by 5 to 50%, 5 to 25%, or 5 to20%. In other embodiments, FEV₁ is increased by 5 to 15% or 5 to 10%. Inother embodiments, FEV₁ is increased by 10 to 50%, 10 to 40%, 10 to 30%or 10 20%. FEV₁ is frequently measured in mL. Accordingly, in someembodiments, FEV₁ is increased by at least 25 mL when compared to FEV₁prior to the treatment. In some embodiments, FEV₁ is increased by 25 to500 mL, 25 to 400, 25 to 300 or 25 to mL. In other embodiments, FEV₁ isincreased by 50 to 500 mL, 50 to 400 mL, 50 to 300 mL, 50 to 200 mL or50 to 100 mL.

In some embodiments, blood oxygen saturation is increased in the subjectcompared to the blood oxygen saturation levels prior to theadministration. In some embodiments, blood oxygen saturation isincreased by at least 1% or by at least 2% for at least 15 days afterthe administration period. In other embodiments, the blood oxygensaturation levels are increased by about 1 to 50%, 1 to 25%, 1 to 20%, 1to 15%, 1 to 10% or 1 to 5%. In other embodiments, the blood oxygensaturation levels are increased by about 2 to 10% or 2 to 5%.

The aforementioned sustained periods of time may be at least 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70 or 75 days after the administrationperiod. In other embodiments, the sustained period of time is at least28, 35, 42, 48 or 56 days after the administration period. In otherembodiments, sustained period of 15 to 75 days, 15 to 35 days, or 20 to35 days. In other embodiments, the sustained period of time is 20 to 30days, 25 to 35 days or 25 to 30 days. In other embodiments, thesustained period of time is about 25, about 26, about 27, about 28,about 29 or about 30 days, or about 28 days, or at least 29 days. Inother embodiments, the sustained period of time during is 25 to 75 days,35 to 75 days, or 45 to 75 days. In other embodiments, the sustainedperiod is 50 to 75 days, 50 to 70 days, 50 to 65 days or 50 to 60 days.In other embodiments, the sustain period is about 50, about 51, about52, about 53, about 54, about 55, about 56, about 57, about 58, about 59or about 60 days, while in other embodiments, the sustained period isabout 56 days.

In some embodiments, the aforementioned methods advantageously provide areduced incidence of pulmonary exacerbations in the patient. The methodalso advantageously increases the length of time to pulmonaryexacerbation. For example, in some embodiments, the length of time topulmonary exacerbation is at least about 20 days. In other embodiments,the length of time is 20 to 100 days. In other embodiments, the lengthof time is 25 to 100 days, 30 to 100 days, 35 to 100 days or 40 to 100days. In other embodiments, the length of time is 25 to 75 days, 30 to75 days, 35 to 75 days or 40 to 75 days. In other embodiments, thelength of time is 30 to 60 days.

In some embodiments, the incidence of rescue treatment is reduced. Inother embodiments, the length of time to rescue treatment is reduced,for example when the patient has a pulmonary infection, the time toanti-infective rescue treatment is reduced. In some embodiments, thelength of time is 20 to 100 days. In other embodiments, the length oftime is 25 to 100 days, 30 to 100 days, 35 to 100 days or 40 to 100days. In other embodiments, the length of time is 25 to 75 days, 30 to75 days, 35 to 75 days or 40 to 75 days. In other embodiments, thelength of time is 30 to 60 days.

In some embodiments, the liposomal amikacin formulation used in theaforementioned methods comprises amikacin and any of the lipidsdescribed above. In some embodiments, the liposomal amikacin formulationcomprises a phospholipid and a sterol, such as DPPC and cholesterol. Inother embodiments, the liposomal amikacin formulation comprises DPPC andcholesterol in about a 2 to 1 ratio by weight. In some embodiments, theliposomal amikacin formulation has a lipid to drug ratio of about 0.5 toabout 1.0, about 0.5 to 0.7, or about 0.6 by weight. In otherembodiments, the liposomal amikacin formulation has a lipid to drugratio of about 0.3 to about 1.0 by weight, while in other embodiments,the lipid to drug ratio is about 0.5 to 0.7 by weight, or about 0.65 byweight. The liposomes in the formulation may have a amend diameter of100 to 1000 nm, 100 to 500 nm, 200 to 500 nm, or about 300 nm. In someembodiments, the total concentration of amikacin in the liposomalamikacin formulation is about 20 to 100 mg/mL, 20 to 90 mg, mL, 30 to 90mg/mL, 30 to 80 mg/mL, or 40 to 80 mg/mL. In other embodiments, theconcentration is about 30, 40, 50, 60, 70, 80 or 90 mg/mL.

In some embodiments, aforementioned method comprises:

administering to the patient an effective dose of a nebulized liposomalamikacin formulation for at least one treatment cycle, wherein:

-   -   the treatment cycle comprises an administration period of about        28 days, followed by an off period of about 28 days;

the effective dose comprises about 280 to about 560 mg of amikacin dailyduring the administration period; and

the liposomal amikacin formulation comprises DPPC and cholesterol inabout a 2:1 ratio, and a lipid to amikacin ratio of about 0.5 to about0.7.

In other embodiments, the method comprises:

administering to the patient an effective dose of a nebulized liposomalamikacin formulation for at least one treatment cycle, wherein:

-   -   the treatment cycle comprises an administration period of about        28 days, followed by an off period of about 56 days;

the effective dose comprises about 280 to about 560 mg of amikacin dailyduring the administration period; and

the liposomal amikacin formulation comprises DPPC and cholesterol inabout a 2:1 ratio, and a lipid to amikacin ratio of about 0.5 to about0.7.

In other embodiments, the present invention relates to a method ofproviding a sustained treatment effect in a subject comprising:administering to the patient an effective dose of a nebulized liposomalamikacin formulation for at least one treatment cycle, wherein: thetreatment cycle comprises an administration period of 15 to 75 days,followed by an off period of 15 to 75 days; and the effective dosecomprises 100 to 2500 mg of amikacin daily during the administrationperiod.

In another embodiment, the present invention relates to a method ofimproving oxygen saturation levels in a patient with a pulmonarycondition comprising: administering to the patient an effective dose ofa nebulized liposomal amikacin formulation for at least one treatmentcycle, wherein: the treatment cycle comprises an administration periodof 15 to 75 days, followed by an off period of 15 to 75 days; and theeffective dose comprises 100 to 2500 mg of amikacin daily during theadministration period.

In another embodiment, the present invention relates to a method ofimproving FEV₁ in a patient with a pulmonary condition comprising:administering to the patient an effective dose of a nebulized liposomalamikacin formulation for at least one treatment cycle, wherein: thetreatment cycle comprises an administration period of 15 to 75 days,followed by an off period of 15 to 75 days; and the effective dosecomprises 100 to 2500 mg of amikacin daily during the administrationperiod.

In another embodiment, the present invention relates to a method ofreducing bacterial density in the lung or sputum of a patient with abacterial pulmonary infection comprising: administering to the patientan effective dose of a nebulized liposomal amikacin formulation for atleast one treatment cycle, wherein: the treatment cycle comprises anadministration period of 15 to 75 days, followed by an off period of 15to 75 days; and the effective dose comprises 100 to 2500 mg of amikacindaily during the administration period, and wherein the bacterialdensity remains reduced for at least 15 days after the last day of theadministration.

EXEMPLIFICATION Introduction to Materials and Methods

Lipid based or liposomal aminoglycoside, such as amikacin, formulationsfor inhalation are sustained-release formulations of aminoglycosidesencapsulated inside nanoscale liposomal carriers designed foradministration via inhalation. Sustained-release targeting of highconcentrations of amikacin in the lungs and biofilm penetrationproperties of these formulations have several advantages over inhalationof the “free” antibiotic, e.g., inhaled tobramycin. Amikacin can beencapsulated in liposomes composed of dipalmitoylphosphatidylcholine(DPPC) and cholesterol, at a targeted lipid-to-drug ratio of about0.6-0.7:1 (w/w). An example of a ˜70 mg/mL liposomal amikacinformulation useful in the aforementioned methods is presented below:

Component Concentration Amikacin¹ ~70 mg/mLDipalmitoylphosphatidylcholine (DPPC) ~30 mg/mL Cholesterol ~15 mg/mL1.5% NaCl QS ¹Added to the formulation as Amikacin sulfate, USP.These formulations can be prepared according to the methods described inU.S. Publication No. 20060073198 or 20080089927, both of which arehereby incorporated by reference.

These formulations have several advantages in treating pulmonaryconditions, for example, CF subjects with chronic infection caused by P.aeruginosa, including:

-   1. The ability to attain a prolonged antibiotic effect of amikacin    in the lung by achieving high concentrations and a prolonged half    life due to sustained release.-   2. The ability to target and increase the effective concentration of    amikacin in the lung with low systemic levels of the aminoglycoside.-   3. The potential to better target bacteria growing in a biofilm as a    result of unique properties of lipid based or liposomal    aminoglycosides.-   4. Additional release of the drug at the site of infection in the    lungs of CF patients, due to targeted action of secreted    phospholipase C and rhamnolipids from bacteria and/or phospholipase    A2 or defensins from activated polymorphonuclear leukocytes.-   5. Amikacin is a semisynthetic aminoglycoside with a unique    resistance to aminoglycoside inactivating enzymes. Consequently,    some P. aeruginosa strains which are resistant to tobramycin will    likely remain susceptible to amikacin.-   6. Amikacin has less binding affinity than other aminoglycosides for    megalin, the transporter responsible for renal cortical    aminoglycoside accumulation, and thus inherently has a lower    potential for nephrotoxicity.-   7. The increase in both the half life, and the area under the    concentration curve (AUC) of lipid based or liposomal amikacin,    along with biofilm penetration should allow for less frequent    administration, enhanced bactericidal activity and reduced potential    for selection of resistant organisms.

Preclinical pharmacokinetics have demonstrated that the AUC (0-48 hr) ofamikacin in the lungs of rats that received a 60 mg/kg dose aerosol ofLiposomal Amikacin was five-fold higher than the AUC of tobramycin inthe lungs of rats that received an equal dose of tobramycin byinhalation. Generally, 10% of the administered antibiotic is depositedin the lungs for rats. Conversely, the AUC of drug in the kidneys ofrats that received an equal dose of tobramycin was significantly higherthan the kidney AUC of rats that received aerosols of LiposomalAmikacin. Additionally, data from 30-day inhalation toxicology studiesin rats and dogs suggest that there will be no safety pharmacologyissues with inhaled Liposomal Amikacin.

In 14 days rat model studies of pseudomonas infection, it was noted that60 mg/kg of Liposomal Amikacin (75 mg/mL) administered every other dayfor 14 days (Q2D×7), which effectively delivered half the cumulativedose of aminoglycoside than the other groups, was as effective as 60mg/kg of Liposomal Amikacin given once per day, and Tobramycin giventwice per day daily for 14 days. With 28 day dosing in this model, therewere equivalent reductions in CFUs in animals receiving LiposomalAmikacin dosed daily at ˜60 mg/kg or dosed every other day at ˜120mg/kg. Liposomal Amikacin administered at 120 mg/kg once a day for 14days was as effective as Tobramycin 60 mg/kg/day (administered twice aday) for 28 days, which suggests a higher AUC and possibly a prolongedpost-antibiotic effect with Liposomal Amikacin at 120 mg/kg dosed onceper day (see Example 3).

The administration of Liposomal Amikacin via inhalation in the animalmodel resulted in increased lung (AUC) above the MIC of the bacteria,and demonstrated sustained therapeutic effect, with a reduced frequency,and duration of dosing as compared to Tobramycin. Importantly, thepreclinical data for Liposomal Amikacin appear supportive of thehypothesis that this specific formulation may be advantageous over otherinhalation products that are hindered by a rapid clearance from lungtissue, necessitating frequent dosing (Geller, Pitlick et al. 2002),which poses a burden for patients and might limit patient compliance.

Additionally, clinical experience demonstrated that nebulized LiposomalAmikacin 50 mg/mL administered as 500 mg once per day for 14 days iswell tolerated, and elicits a clinically relevant effect on pulmonaryfunction and decrease in P. aeruginosa density in CF patients. Also,evaluation of the PK data indicates the systemic exposure to LiposomalAmikacin, even at the 500 mg dose, is very low. By either Cmax or AUC ormg of aminoglycoside which is recovered in the urine, the observedsystemic exposure to amikacin, associated with Liposomal Amikacin, givenby inhalation is approximately ⅕ to ¼ the exposure seen with 600 mg/d ofTOBI and is less than 1/200 compared to normal parenteral doses ofAmikacin. The data further indicate high levels of Amikacin are achievedin the sputum. Median AUC values for sputum were 290 and 980 foldgreater than the median AUC values for serum on day 1 and day 14respectively.

Inhaled liposomal amikacin maintains prolonged targeted lung exposuresand enhance the uptake of drug to the site of infection. Using data froma human clinical Phase 1b/2a study in which CF patients who werechronically infected with P. aeruginosa received multiple doses ofLiposomal Amikacin 50 mg/ml, the objectives of the analyses describedherein were three-fold: (1) to use population pharmacokinetic (PK)modeling to characterize amikacin systemic exposure, includingapproximate systemic bioavailability; (2) to characterize thedisposition of liposomal amikacin in sputum; and 3) to characterize thepharmacokinetic-pharmacodynamic (PK-PD) relationship between change inforced expiratory volume in one second (FEV₁), change in percentpredicted forced expiratory volume in one second (FEV₁% predicted),forced expired flow between 25-75% of forced vital capacity (FEF₂₅₋₇₅%),and forced vital capacity (FVC), in P. aeruginosa colony forming units(CFU) relative to baseline at Days 7 and 14, and amikacin exposure.

Preclinical Studies with Liposomal Amikacin

Several preclinical studies were conducted with the 20 and 50 mg/mLformulations. Anti-pseudomonas activity of Liposomal Amikacin in invitro and in vivo models was demonstrated. Additionally, studiesconfirmed that virulence factors secreted by Pseudomonas facilitate thefurther release of amikacin from the liposomes, and characterized thedeposition and sustained release of amikacin in the lungs of rats, anddogs. The safety of a 30 day administration of Liposomal Amikacin in twospecies was also established.

Nonclinical pharmacokinetics have demonstrated that the AUC (0-48 hr) ofamikacin in the lungs of rats that received a 60 mg/kg dose of LiposomalAmikacin via nebulization, was five-fold higher than the AUC oftobramycin in the lungs of rats that received an equal dose oftobramycin by inhalation. High levels of amikacin were sustained in thelung (>250 μg/mL through 150 hr), suggesting a depot effect. Incontrast, lung levels of tobramycin were undetectable within 6 hours ofcessation of administration. Conversely, the AUC of drug in the kidneysof rats that received an equal dose of tobramycin was significantlyhigher than the AUC of rats that received aerosols of LiposomalAmikacin. There were no significant differences in the AUC ofaminoglycosides in the serum and urine of the animals; serum levels wereundetectable after 24 hr. This profile supports the intended sustainedrelease and depot effect of amikacin in the lung followingadministration of nebulized Liposomal Amikacin, potentially representingan enhanced efficacy profile. These data for Liposomal Amikacin appearsupportive of the hypothesis that this specific formulation may beadvantageous over other inhalation products that are hindered by a rapidclearance from lung tissue, necessitating frequent dosing (Geller,Pitlick et al. 2002), and placing a burden on patients. Additionally,toxicokinetic data from 30-day inhalation GLP toxicology studies in ratsand dogs showed that there is a 15 fold increase in lung deposition ofamikacin dogs as compared to the free amikacin treated group, withcomparable plasma and urine levels, indicating high lung concentrationswith low systemic exposure.

The pharmacodynamic effect of Liposomal Amikacin was evaluated in vivoin a rat model of chronic pulmonary infection with Pseudomonas (Cash,Woods et al. 1979). In a 14 days Pseudomonas infection model, 60 mg/kgof Liposomal Amikacin (75 mg/mL) was administered every other day for 14days (Q2D×7). This regimen was as effective as 60 mg/kg of LiposomalAmikacin (given once per day for 14 days), and tobramycin (given twiceper day for 14 days). When dosing was extended to 28 days, there wereequivalent reductions in CFUs for animals receiving Liposomal Amikacindosed daily at ˜60 mg/kg or dosed every other day at ˜120 mg/kg. Also,in this experiment, Liposomal Amikacin administered at 120 mg/kg once aday for 14 days was as effective as tobramycin 60 mg/kg/day(administered twice a day) for 28 days. This indicated a higher AUC anda prolonged post-antibiotic effect with Liposomal Amikacin at 120 mg/kgdosed once per day. The preclinical pharmacodynamic data were thusconsistent with a sustained antimicrobial benefit enhanced by thesite-specific delivery of drug to the lungs via inhalation.

Thus, administration of Liposomal Amikacin via inhalation resulted inincreased lung concentrations (AUC) several fold above the MIC of thebacteria, with the potential to provide a sustained therapeutic effectwith a reduced frequency and duration of dosing, particularly ascompared to Tobramycin.

Example 1 Phase 1b/2a Study

Data used for this population PK analysis were obtained from two humanclinical Phase 1b/2a studies in which CF patients, chronically infectedwith P. aeruginosa, were administered a total of 500 mg of LiposomalAmikacin daily (in two 20 minute sessions with a 5 minute rest period inbetween) for 14 days.

Amikacin serum samples were obtained pre-dose, and 1, 2, 4, 6, 8, 12 and24 hours post-dose on Days 1 and 14, while urine samples were collectedover 6 hour intervals on Day 1 and Day 14 for a period of 24 hours.Sputum samples were also collected on Day 1 and Day 14, soon after thedose was administered, between 4 and 6 hours after dosing and prior todose administration on the following day, as well as on Days 14, 21, and28. Serum, sputum and urine samples were assayed for amikacin usingLiquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS).

Pulmonary function tests (PFT) were carried out during screening fromDay −14 to 0) and at baseline (i.e., prior to dose administration onDay 1) and on Day 1, 7, 14, 21, 28, 35, and 42. Sputum samples formicrobiology were also collected at baseline and on each of these days.Additional PFTs were carried out 1.5 hours and 3 hours post-dose on Day1 and Day 14.

Pharmacokinetic Analysis

The data were fit by candidate PK models, using Monte Carlo ParametricExpectation Maximization (MC-PEM), as is implemented in S-ADAPT 1.53,initially fitting the plasma concentrations, then co-modeling the serumand urine data. Model discrimination was based on the fit of the dataand change in objective function. The 24 hour area under the curve (AUC)at steady state for serum amikacin values were calculated using thepost-hoc parameter estimates from the final population PK model.Covariate relationships between patient demographics and individualpost-hoc parameters were assessed first graphically, then by means ofstatistical models created using SYSTAT® 11 (SYSTAT Software, Inc.,Richmond, Calif.). Sputum AUC values from 0 to 24 hours on Day 1 and Day14 were obtained using the linear trapezoidal rule.

Dependent variables for the PK-PD analysis included the change in PFTvalues for FEV₁, FEV₁% predicted, FEF_(25-75%) and FVC, on Day 7 and 14relative to baseline (prior to dose administration on Day 1) and thechange in log₁₀ CFU on each of these days relative to baseline.Independent variables evaluated included the ratio of the average 24hour AUC for serum and sputum to the baseline minimum inhibitoryconcentration (MIC), AUC:MIC ratio for P. aeruginosa. The average 24hour serum and sputum AUC was computed by taking the average of the Day1 and Day 14 AUC values.

Using a one-sample t-test, the statistical significance of mean changesfrom baseline for each of the above-described dependent variables wasassessed. Using Spearman's rank correlation (r_(s)), the direction andstrength of the relationship between each of the dependent variables andAUC:MIC ratio for serum and sputum was assessed. The direction andstrength of the relationship between change in each of the PFT valuesfrom baseline and change in log₁₀ CFU from baseline were also assessed.

Results

A total of 24 patients completed the two studies with 13 patients fromStudy 1 and 11 patients from Study 2. The median (min, max) age of allthe patients was 23.7 (14, 38) years with a median (range) creatinineclearance (CrCL) at baseline of 126 (76.8, 173) mL/min/1.73 m².

The most robust fit to the serum concentration data was obtained using atwo-compartment model (one absorption site, the lung, and the centralcompartment) with zero-order drug input into the lungs, a first-orderprocess from lungs to the central compartment and linear elimination.Allowing inter-occasional variation on apparent total clearance (CLt/F)and apparent central volume of distribution (Vc/F) between Day 1 and Day14 improved the objective function statistically. Urine data was modeledby fitting the amounts of amikacin recovered in the collectionintervals, as a function of serum concentrations and renal clearance(CLr). Table 1 is a summary of the fitted PK parameter values.

TABLE 1 Structural population pharmacokinetic model for liposomalamikacin for inhalation with inter-occasional variability - Parameterestimates and standard errors. Inter-individual Population meanvariability (% CV) Final Final Parameter estimate % SE estimate % SECLt/F Day 1 (L/hr) 68.4 10.3 48.7 29.9 Vc/F Day 1 (L) 286 12.3 59.0 29.7ka (hr⁻¹) 3.34 32.5 99.8 50.5 CLr (L/hr) 3.40 15.4 63.9 36.7 CLt/F Day14 (L/hr) 45.2 8.01 37.1 30.7 Vc/F Day 14 (L) 250 8.51 27.0 30.8SDint_(Serum) 0.05 6.02 SDslp_(Urine) 0.70 9.16 SDint_(Urine) 0.03Minimum value of the objective function = −258.6

The goodness of fit for observed versus Bayesian post-hoc individualfitted serum concentration data was excellent, with an overall r² of0.98.

The AUC values for the serum and sputum data are shown in Tables 2 and3, respectively. Median AUC values for sputum were 286 and 978 foldgreater than the median AUC values for serum on Day 1 and Day 14,respectively. As evidenced by the higher CV % values, greatervariability was evident in sputum (117% on Day 1 and 91.2% on Day 14)compared to serum AUC (51.9% on Day 1 and 42.4% on Day 14) values.

TABLE 2 Summary of serum AUC values¹ - All patients Study Day N Mean SDMin Median Max Day 1 24 8.27 4.29 3.67 6.88 20.1 Day 14 24 12.0 5.085.65 10.8 30.1 ¹AUC values in mcg/mL · hr

TABLE 3 Summary of sputum AUC values¹ - All patients Study Day N Mean SDMin Median Max Day 1 20 3830 4500 78.70 1970 17200 Day 14 19 12500 114001740 10578 50000 ¹AUC values are in mcg/mL · hr

Serum (r²=0.98) and urine (r²=0.38) concentrations were well andmodestly fit by model, respectively. On Day 7, 14 and 21, the observedchange for FEF_(25-75%) was 0.49 (p<0.001), 0.42 (p=0.02) and 0.34 L/sec(p=0.04), respectively. On Day 7 and 14, the observed change for FEV₁was 0.24 (p=0.002) and 0.13 L (p=0.10), respectively, and was 7.49(p<0.001) and 4.38 L/sec (p=0.03) for FEV₁% predicted. Significantrelationships (p≦0.05) between log₁₀ CFU and serum AUC:MIC ratio, andbetween changes in log₁₀ CFU and FEV₁, FEV₁% predicted and FVC wereidentified.

Baseline and Day 14 PFT data were available for all 24 patients and forPFTs carried out on Day 7 and 21, such data were available for 23patients. Microbiology data were available for all 24 patients. SinceMIC values collected prior to dosing on Day 1 for Study 2 were notreported, the screening MIC values as well as CFU counts were used asbaseline values.

Using a one-sample t-test, the statistical significance of mean changesfrom baseline for each of the above-described dependent variables wasassessed. Using Spearman's rank correlation (r_(s)), the direction andstrength of the relationship between each of the dependent variables andAUC:MIC ratio for serum and sputum was assessed.

Mean changes in PFT values on Day 7 relative to baseline werestatistically significant for all PFT endpoints. Mean changes in FEV₁%predicted and FEF_(25-75%) on Day 14 relative to baseline were alsostatistically significant (p=0.029 and p=0.016, respectively). By Day21, mean change in FEF_(25-75%) relative to baseline was the single PFTthat remained statistically significant (p=0.036). Regardless of thestudy day considered, mean change in log₁₀ CFU from baseline was notstatistically significant.

As shown in Table 4, correlations between change in PFT values frombaseline and either sputum or serum AUC:MIC ratio were not statisticallysignificant, regardless of whether changes on Day 7 or 14 wereevaluated. As shown in Table 5, the correlation between change in log₁₀CFU from baseline and serum AUC:MIC ratio was statistically significantfor both Day 7 or 14. Increasing serum AUC:MIC ratios were associatedwith larger decreases in log₁₀ CFU on Day 7 (r_(s)=−0.46, p=0.048) and14 (r_(s)=−0.45, p=0.048) relative to baseline.

Correlations between change in both PFT value and log₁₀ CFU on Day 7 and14 relative to baseline were statistically significant for FEV₁, FEV₁%predicted, and FVC (p<0.05).

TABLE 4 Relationship between change in pulmonary function test valuesfrom baseline and AUC:MIC ratio for serum and sputum - All patientsChange in PFT values from baseline Study Day Spearman's FEV₁ % AUC:MICrank correlation FEV₁ predicted FEF_(25-75%) FVC Day 7 serum r_(s) ²0.072 0.0066 <0.0001 0.021 p value 0.21 0.71 0.97 0.51 Day 14 serumr_(s) ² 0.046 0.0073 0.00018 0.0012 p value 0.31 0.69 0.95 0.87 Day 7sputum r_(s) ² 0.033 0.040 0.0085 0.19 p value 0.46 0.41 0.71 0.06 Day14 sputum r_(s) ² 0.025 0.052 0.0053 0.06 p value 0.51 0.35 0.77 0.31

TABLE 5 Relationship between change in log₁₀ CFU and AUC:MIC ratio forserum and sputum - All patients Study Day Spearman's log₁₀ AUC:MIC rankcorrelation CFU Day 7 serum r_(s) ² 0.21 p value 0.048 Day 14 serumr_(s) ² 0.20 p value 0.048 Day 7 sputum r_(s) ² 0.017 p value 0.64 Day14 sputum r_(s) ² 0.0031 p value 0.84

While mean change in log₁₀ CFU of P. aeruginosa from baseline on bothDay 7 and 14 was not statistically significant, the correlation betweenchange in log₁₀ CFU from baseline at both of these time points and serumAUC:MIC ratio was statistically significant; increases in serum AUC:MICratio were associated with decreases in log₁₀ CFU. In contrast, thisrelationship did not hold with sputum AUC:MIC and confirms the largevariability in sputum kinetics of Liposomal Amikacin, that is also shownwith TOBI (Geller, Pitlick et al. 2002).

The significant relationships between changes in log₁₀ CFU and serumAUC:MIC ratio, and between changes in PFT values and log₁₀ CFU, and thelack of significant decrease in log₁₀ CFU of P. aeruginosa during thetwo weeks of treatment with liposomal amikacin for inhalation suggeststhat higher doses may be required to be more reliably effective in alarge patient population.

Summary of the Phase 1a/2b Study

Two Phase 1b/2a studies using the Liposomal Amikacin 50 mg/mL have beencompleted. The two studies were similar in design. A total of 24 CFpatients (with FEV₁≧40% of predicted) received 500 mg Liposomal Amikacindaily for 14 days. The drug was administered using a PARI LC Starnebulizer, over a period of two 20-minute inhalation sessions with a 5minute rest period between sessions. There were 13 patients enrolled inStudy 1 and 11 patients in Study 2. Patient demographics were similar,with the exception of Pseudomonas MICs at baseline. In Study 1, the meanMIC (μg/mL) was 8 (range 1.5-16) and in Study 2, the mean MIC was 41μg/mL (range 8-192). The patients enrolled in Study 2 had priorexperience with inhalation antibiotics, and per protocol, were permittedto resume treatment with TOBI®/Colistin after Day 28 of the study. Thepatients in Study 1 were naïve to inhalation antibiotics, and did notreceive additional inhalation antibiotics during the follow-up period.The 500 mg dose of Liposomal Amikacin (50 mg/mL) was well tolerated, andin select patients improved pulmonary function and decreased the densityof P. aeruginosa in sputum. The details of patient demographics forStudies 1 and 2 (combined) are shown in Table 6.

TABLE 6 Patient demographics in studies 1 and 2. Variable Mean SD MedianMin Max Age 23.7 6.96 22.5 14.0 38.0 Weight (kg) 59.1 13.0 58.6 43.499.6 Height (cm) 168 8.10 168 155 194 IBW (kg) 61.4 8.99 60.0 47.9 87.7CrCL (mL/min) 125 20.9 126 76.8 173All efficacy analyses in these human clinical Phase 1b/2a studies wereexploratory in nature. The efficacy endpoints included:Change from Baseline in density of P. aeruginosa (log₁₀ CFU/g) insputum;Change from Baseline in pulmonary function tests (FEV₁, FEV₁% predicted,FVC, and FEF_((25-75%)).Changes in P. aeruginosa sputum density, FEV₁, and FEV₁% predicted atDay 14 were identified as the primary efficacy endpoints.

Quantitative culture of sputum samples and subsequent amikacinsusceptibility testing of each morphologically distinct P. aeruginosawere performed. The MIC of amikacin for the isolates with the highestMIC cultured from each subject at screening and Day 14 was documented.The density (CFU per gram of sputum) of P. aeruginosa in sputum wascalculated as the log₁₀ value for the sum of all morphotypes.

A summary of the baseline characteristics for the combined population(n=24) are shown in Table 7.

TABLE 7 Baseline measurements for patients in Studies 1 and 2. VariableMean SD Median Min Max FEV1 (L) 2.38 1.07 2.18 1.15 6.10 Predicted FEV1% (L/sec) 65.5 18.9 62.5 40.0 119 FEF25-75 (L/sec) 1.71 1.26 1.49 0.555.50 FVC (L) 3.32 0.92 3.27 1.67 5.28 Log10 CFU Count 7.05 1.3 7.3 3.518.98 MIC (mcg/mL) 35 56 10 2 192

Study 1:

In this study CF patients infected with P. aeruginosa isolates sensitiveto amikacin (amikacin MIC<64 μg/mL), and those subjects naïve to inhaledantibiotics were enrolled. Administration of Liposomal Amikacin 500 mgonce daily for 2 weeks showed a mean change in log sum of counts of P.aeruginosa from baseline to Day 14 of 1.09 (n=13; 95% confidenceinterval, 2.09 to 0.09). The reductions in counts were observed in 9 ofthe 13 subjects. Treatment with Liposomal Amikacin did not result inselection of resistant strains of P. aeruginosa. The mean P. aeruginosaamikacin MIC was 8.04 μg/mL at Day 0 and 30.79 μg/mL at Day 14. On Day14, a single isolate in one subject had a non sensitive MIC (>256μg/mL); all other Day 14 isolates were sensitive to amikacin. No humanwas hospitalized or received intravenous anti-Pseudomonas antibiotics.Additionally, there was improvement in lung function as measured by anincrease in FEV₁ from baseline to Day 14 of +260 mL (n=13; 95%confidence interval, +30 mL to +500 mL). The corresponding change inFEV1% predicted from baseline to Day 14 was +7.32%. Increases in FEV1were observed in 9 of the 13 subjects. Also noted were increases inFEF_((25-75%)) (mean: 570 mL) and FVC (mean: 180 mL).

Study 2:

Study 2 was conducted in a population of CF patients who were infectedwith P. aeruginosa, and were inhalation antibiotic treatmentexperienced. In these patients, the administration of Liposomal Amikacin500 mg q.d. for 2 weeks did not show any significant change in P.aeruginosa density during the study (p-values >0.297 for change from Day1). The proportion of patients with mucoid P. aeruginosa remainedconstant throughout the study. No statistically significant changes inFEV₁, FEV₁% predicted, FVC, and FEF_((25-75%)) were observed afteradministration of Liposomal Amikacin 500 mg. Nevertheless, trendssuggesting improvement in FEV₁% predicted, FVC, and FEF_((25-75%)) wereobserved at Day 7, Day 14 (end of treatment), and Day 15.

Integrated Efficacy Summary: Studies 1 and 2

Data from the combined population of 24 patients in studies 1 and 2 aresummarized below in Tables 8, 9, 10, and 11. The microbiologic end-pointof change in log CFU of P. aeruginosa, demonstrated a reduction inbacterial density in the combined population, but this did not achievestatistical significance. But, when data were analyzed from theinhalation antibiotic naïve patients (study 1), a statisticallysignificant reduction in CFU was observed at end of treatment. Factorsthat might explain this effect are the inherent variability in sputumsamples, the inter-laboratory variability in methodology, and reportingof quantitative microbiology, and the enrollment of patients with higherMICs (including resistant isolates) in study 2. All of the above arefurther compounded by the small sample size of each study.

Assessment of clinical benefit by measurement of pulmonary functiontests showed a statistically significant improvement in lung function asmeasured by an increase in FEV₁ from baseline to Day 7 of +240 mL (n=23;p-value 0.0024). The effect at day 14 was a 126 mL increase frombaseline in FEV1, which was not statistically significant. Acorresponding statistically significant increase in FEV1% predicted frombaseline to Day 7 was +7.49% (n=24; p-value 0.0002), and at Day 14 was+4.37% (n=24; p-value 0.0285). The improvement in lung function was alsonoted with the assessment of small airways as measured by FEF_((25-75%))at day 7, an increase in +494 mL (n=23; p-value 0.001), and at Day 14,+423 mL (n=24; p-value 0.0162). These data support a clinicallymeaningful improvement in lung function in CF patients with chronicPseudomonas infection who have received a 14 day course of treatmentwith Liposomal Amikacin.

TABLE 8 Change in FEV from baseline at various times in all patients.Time Point N Mean CV p-value Day 7 (pre-dose) 23 0.24 1.4 0.0024 Day 14(pre-dose) 24 0.126 2.86 0.1006 Day 21 23 0.073 4.91 0.3397

TABLE 9 Change in % predicted FEV from baseline at various times in allpatients. Time Point N Mean CV p-value Day 7 (pre-dose) 23 7.491 1.090.0002 Day 14 (pre-dose) 24 4.379 2.10 0.0285 Day 21 23 2.713 3.250.1544

TABLE 10 Change in FEF₂₅₋₇₅ from baseline at various times in allpatients. Time Point N Mean CV p-value Day 7 (pre-dose) 23 0.494 1.260.001 Day 14 (pre-dose) 24 0.423 1.89 0.0162 Day 21 23 0.338 2.15 0.0361

TABLE 11 Change in CFU from baseline at various times in all patients.Time Point N Mean CV p-value Day 7 19 −0.154 −7.37 0.5616 Day 14 20−0.315 −4.42 0.3242 Day 21 20 0.24 5.4 0.4182

Example 2 Phase 1 Clinical Study

Two Phase 1 single dose clinical studies were completed with 20 and 50mg/mL formulations of Liposomal Amikacin in healthy volunteers and in CFpatients, respectively. Six healthy volunteers received a single dose of120 mg of Liposomal Amikacin and tolerated it well, and exhibitedprolonged retention of the radiolabeled liposomes in the lungs, with ameasured half-life of 46 hours.

Liposomal Amikacin was administered to CF subjects with chronic P.aeruginosa infections in a human clinical Phase I study (Study 3).Single doses of 90 mg (n=6), 270 mg (n=6), or 500 mg (n=4) wereadministered to CF subjects to evaluate the safety, tolerability andpharmacokinetics of liposomal amikacin for inhalation. A total of 24patient dosing sessions of a single dose administration of LiposomalAmikacin or placebo by inhalation via the Pari LC Star nebulizer wereevaluated. Two serious adverse events were reported (both occurring inplacebo group). Both events recovered without sequelae. A total of 41adverse events (AEs) were experienced by 17 of the 24-patient sessionsdosed (71%) during the trial. Of the AEs reported, 10 of the 16 patients(62.5%) who reported adverse events were in the active group and 7 ofthe 8 patients (87.5%) were in the placebo group. Headache was the mostcommon AE reported in the active group and no patients were discontinuedfrom the study due to AEs. Liposomal Amikacin was well tolerated andsafe up to a single dose of 500 mg administered via inhalation.

Additionally, the PK data confirm minimal systemic drug levels, and highsputum levels of drug, and pharmacodynamic modeling estimates longelimination half life presumably due to slow release from liposomes.

Example 3 Phase 2 Clinical Study

The study design is summarized in FIG. 4. Patients included in the studywere CF patients greater than or equal to six years in age with chronicP. aeruginosa infections. Patients were off inhaled antibiotics for 28prior to beginning the study. Patients were stratified by baselineFEV1(% pred) and randomized 2:1 to Arikace™ or placebo (1.5% NaCl).Cohort1 received 280 mg and Cohort2, 560 mg of active drug or placebofor 28 d by inhalation with PARIeFlow® nebulizer, and were followed for28 d during which no inhaled antibiotics were administered. Safety,pharmacokinetics, Pa sputum density, Quality of Life (CFQ-R) andexacerbation rate were evaluated weekly during the study period of 56days.

In summary, daily administration of 280 mg and 560 mg liposomal amikacinfor 28 days appeared safe and well-tolerated. Administration ofliposomal amikacin at 280 mg and 560 mg for 28 days results in adose-dependent improvement in lung function, which is sustained at leastfor 28 days after the completion of the dosing. The patients receivingliposomal amikacin experienced fewer pulmonary exacerbations (7.14%)compared to those receiving a placebo (18.18%). Additionally, the timeto exacerbation was prolonged in the amikacin groups (41 days) comparedto the placebo (19 days). The groups receiving amikacin experienced nopulmonary exacerbations during the 28 day treatment period. Patientsreceiving liposomal amikacin demonstrated greater clinical benefitcompared to the placebo group as measured by improvement in the qualityof life CFQR-respiratory scale.

FIGS. 5 and 6 depict graphs showing the change in oxygen saturation frombaseline in pediatric patients (ages 6 to 12) compared to placebo. Theresults demonstrate an improvement in oxygen saturation beginning duringthe 28 day treatment period and continuing beyond the treatment period.A similar improvement in oxygen saturation was observed in patients overthe age of 12 as well.

FIGS. 7a and 7b depict the change in lung function as measured by theforced expiratory volume (FEV₁) in the placebo group and the amikacingroup, respectively, broken down by age groups. Patients in the placebogroup show an overall decrease in FEV₁ by day 56, while patientsreceiving liposomal amikacin consistently demonstrated an increase inFEV₁ both during and up to 28 days after treatment. The placebo grouphad the following change in lung function values (measured in mL):

TABLE 12 Change in FEV₁ in placebo group. age Day 7 Day 14 Day 21 Day 28Day 35 Day 56  6-12 −79 −88 (117) 26 0 (61) −6 −4 13-18 87  2 (80) 25 26(149) 25 −65 18+ 102 −22 (150) 46 36 (135) −24 −56The amikacin group had the following change in lung function values(mL):

TABLE 13 Change in FEV1 in liposomal amikacin treated group. age Day 7Day 14 Day 21 Day 28 Day 35 Day 56  6-12 173 232(117) 138 154 (165) 110178 13-18 136 133 (157) 143 158 (153) 79 44 18+ 103  94 (107) 68 46 (95)29 55

A comparison of the change in FEV₁ from baseline (measured in mL) forall patients in the 560 mg, 280 mg and placebo groups is depicted inFIG. 8. Again, the data demonstrates a sustained effect lasting as longas day 56 in patients receiving liposomal amikacin, where the effect iseven more pronounced in the 560 mg group compared to the 280 mg group.FIG. 9 represents the change from baseline as a percentage. FEV1increased significantly in the 560 mg group, with a sustained treatmenteffect of a 224 mL (a 17.6%) increase compared to the placebo at day 56.

The data from the study also demonstrated a significant reduction inCFU's in patients receiving liposomal amikacin compared to the placebo,and this reduction was sustained at least to day 35. The reduction inCFU was more pronounced for the group receiving 560 mg of amikacincompared to the 280 mg group, as seen in FIG. 10. FIG. 11 depicts theLog CFU change for mucoid strains. These results demonstrate that P.aeruginosa density was reduced, as measured by log CFU, in the groupsreceiving liposomal amikacin, compared to placebo, and this effect wassustained at least to day 35 of the study. Patients with mucoid strainsof P. aeruginosa also were susceptible to treatment with liposomalamikacin. A 1.2 log CFU reduction was seen in the 280 mg group, and a2.0 log reduction in the 560 mg group. The reduction was sustained atday 35 of the 560 mg group with a 1.8 log CFU reduction, while thereduction was sustained with a log 0.4 CFU reduction in the 280 mggroup.

The pharmacokinetic data revealed high levels of amikacin in the sputumof patients receiving liposomal amikacin, with the mean Cmax (CV) of3496 (0.973) mcg/g. The mean area under the curve (AUC) value was 13,120(1.63) mcg/g*hr for the 280 mg group, while the mean AUC was 22,445(0.831) mcg/g*hr). The serum pharmacokinetic data, on the other hand,demonstrated low systemic exposure to amikacin, with the Cmax mean (SD)of 2.27 (1.58) mcg/mL.

Patients receiving liposomal amikacin also had a reduced frequency andtime to pulmonary exacerbation. Table 14:

TABLE 14 Pulmonary Exacerbations. Arikace Placebo Patients 3/42 (7.1%)3/22 (13.6%) Time to exacerbation (days) 40.6* 19.3 *No exacerbationduring treatment period.As seen in Table 14, the percentage of exacerbations in patients treatedwith liposomal amikacin (including both the 280 mg and 560 mg groups)was lower compared to the placebo group. Moreover, the time toexacerbation was much longer in patients receiving liposomal amikacin(40.6 days) compared to 19.3 days in the placebo group.

Anti-Pseudomonal rescue treatments was also reduced in patientsreceiving inhaled liposomal amikacin, compared to the placebo group, asseen in Table 15.

TABLE 15 Anti-Pseudomonal rescue treatment. Arikace Placebo Patients4/42 (9.5%) 3/22 (13.6%) Time to exacerbation (days) 43.0* 21.3 *Norescue during treatment period.As seen in table 15, a lower percentage of patients receiving inhaledliposomal amikacin required anti-Psuedomonal rescue treatment, comparedto the placebo group.Additionally, the time before rescue treatment was needed was reduced inthe liposomal amikacin patients (43.0 days) compared to the placebogroup (21.3 days).

Example 4 Nebulization of Liposomal Amikacin

The aerosol properties of Liposomal Amikacin produced from the eFlow 40L are shown in Table 15. When compared to nebulizate generated from theLC Star, the mass median aerodynamic diameter (MMAD) values for theeFlow are ˜0.5 μm larger. The actual size dependent mass distributionsfrom both ACI (with eFlow) and NGI (with LC Star) cascade impactors fornebulized Liposomal Amikacin are shown in FIG. 1. Aerosol from theeFlow/ACI measurements was slightly narrower in size distribution thanthat from the LC Star/NGI. This difference is reflected in the lowermean geometric standard deviation (GSD) (1.66 versus 1.99) which is ameasure of the width of the distribution around the MMAD, see values inTable 14. This narrower distribution offsets any potential effect of alarger MMAD and therefore, the amount of nebulized drug in therespirable range (<5 μm droplet size) is comparable for both eFlow andLC Star.

TABLE 14 Properties of Liposomal Amikacin Nebulized with the eFlow andLC Star Nebulizers. Aerosol Droplet Properties Percent AssociatedCascade Amikacin Impactor MMAD GSD Respirable Pre- Post- Nebulizer Used(μm) (unitless) Fraction* Nebulization Nebulization eFlow Andersen 368 ±0.26 1.66 ± 0.07 72.9 ± 5.5 96.3 ± 2.1% 66.3 ± 5.8% LC Star NGI 3.18 ±0.18 1.99 ± 0.05 74.5 ± 2.6 96.3 ± 2.1% 62.1 ± 7.4% The Andersen cascadeimpactor was used at a flow rate of 28.3 L/min, 18° C., and 50%humidity. The NGI impactor was used at a flow rate of 15 L/min and 5° C.to achieve >60% humidity. *Percent mass of the nominal drug dose that isless than 5 μm in diameter.

Example 3 Effect of Liposomal Amikacin on P. aeruginosa Lung Infectionsin Rat

The efficacy of Liposomal Amikacin for Inhalation, Liposomal Amikacinwas studied using a model for chronic pulmonary infection (Cash, Woodset al. 1979) where P. aeruginosa, embedded in an agarose bead matrix,was instilled in the trachea of rats. This mucoid Pseudomonas animalmodel was developed to resemble the chronic Pseudomonas infections seenin CF patients (Cantin and Woods 1999). Rat lungs were inoculated with10⁴ CFUs of a mucoid P. aeruginosa strain (mucoid strain 3064)originally isolated from a CF patient. Three days later, 60 mg/kgLiposomal Amikacin (75 mg/mL) was administered by inhalation once dailyfor 14 doses (Q1D×14) or every other day for 7 doses (Q2D×7) (6 mg/kgper dose). For comparison, tobramycin was administered by inhalation BIDfor 14 days (30 mg/kg per dose for a total of 60 mg/kg daily). There wasa significant reduction in bacterial density in all three treatmentgroups as compared to the saline control (see FIG. 2). There were nosignificant differences in the reduction of log₁₀ CFU/lung between thethree treatment groups of rats. It should be noted that LiposomalAmikacin (75 mg/mL) administered every other day for 14 days (Q2D×7),which effectively delivered half the cumulative dose of aminoglycoside,was as effective as the daily dosing regimen in this model.

As shown in FIG. 3 when dosing was extended in this model to 28 days,there were equivalent reductions in CFUs for animals receiving LiposomalAmikacin dosed daily at ˜60 mg/kg or dosed every other day at ˜120mg/kg. Nevertheless, this was only seen as statistically significant forthe latter group when compared to animals that received 1.5% saline onthe same schedules (p=0.24 and 0.03, respectively). In both cases, therewas a significant number of animals in the saline control groups thatalso experienced 2 log reductions in the CFUs. The longer duration (post14 days) of saline inhalation treatment seemed to enhance thespontaneous ability of rats to clear their lungs of infection andpresumably the agar beads which maintain the chronic infectioncondition. Rats that received Liposomal Amikacin ˜120 mg/kg daily for 14days, were observed for another 14 days, and then euthanized on day 35.Lungs of these animals had bacteria below the limit of detection, as wasthe case in the group that received tobramycin 60 mg/kg (given twice perday) daily for 28 days, and then euthanized. Data indicate that in thisexperiment, Liposomal Amikacin administered at 120 mg/kg once a day for14 days was as effective as tobramycin 60 mg/kg/day (administered twicea day) for 28 days. This result suggests a higher AUC and possibly aprolonged post-antibiotic effect with Liposomal Amikacin at 120 mg/kg.

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INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. published patent applications citedherein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of treating a pulmonary disorder in a patient comprisingadministering to the patient an effective dose of a nebulized liposomalamikacin formulation for at least one treatment cycle, wherein: thelipid component of the liposomal amikacin formulation comprises aphospholipid and cholesterol, the treatment cycle comprises anadministration period of 15 to 75 days, followed by an off period of 15to 75 days; and the effective dose comprises 100 to 1000 mg of amikacindaily during the administration period.
 2. The method of claim 1,wherein the treatment cycle is followed at least twice.
 3. The method ofclaim 1, wherein the administration period is 15 to 35 days or about 20to 35 days. 4.-9. (canceled)
 10. The method of claim 1, wherein the offperiod is 35 to 75 days. 11.-16. (canceled)
 17. The method of claim 1wherein the effective dose is about 280 to about 560 mg of amikacin. 18.(canceled)
 19. The method of claim 1, wherein the effective dose isabout 510 to about 610 mg. 20.-22. (canceled)
 23. The method of claim 1,wherein the pulmonary disorder is selected from the group consisting ofchronic obstructive pulmonary disease, bronchiectasis, pulmonaryinfection, cystic fibrosis, alpha-1-antitrypsin enzyme deficiency and acombination thereof.
 24. The method of claim 23, wherein the pulmonarycondition is a bacterial pulmonary infection.
 25. The method of claim24, wherein the pulmonary infection is a P. aeruginosa infection. 26.The method of claim 23, wherein the pulmonary condition isbronchiectasis.
 27. The method of claim 1, wherein the patient has aserum C_(max) of amikacin of less than about 10 mcg/mL during theadministration period.
 28. The method of claim 1, wherein the patienthas a sputum C_(max) of amikacin of at least 1000 mcg per gram ofsputum.
 29. The method of claim 24, wherein the sputum C_(max) ofamikacin is at least 1000 mcg per gram of sputum during theadministration.
 30. The method of claim 24, wherein the sputum C_(max)of amikacin is at least 1000 mcg per gram of sputum for at least 15 daysafter the administration.
 31. The method of claim 24, wherein thepatient has a reduction in log₁₀ CFU of the bacterial infection in thelungs of at least 0.5 for at least 15 days after the administrationperiod ends.
 32. The method of claim 31, wherein the reduction in thelog₁₀ CFU is at least 1.0.
 33. The method of claim 1, wherein thepatient experiences an improvement in lung function for at least 15 daysafter the administration period ends.
 34. The method of claim 33,wherein the improvement comprises an increase in FEV₁, an increase inblood oxygen saturation, or both.
 35. The method of claim 34, whereinthe patient has an FEV₁ that is increased by at least 5% over FEV₁ priorto the treatment cycle.
 36. The method of claim 34, wherein FEV₁ isincreased by about 5 to about 50%.
 37. The method of claim 34, whereinFEV₁ is increased by about 25 to about 500 mL over FEV₁ prior to thetreatment cycle.
 38. The method of claim 34, wherein blood oxygensaturation is increased by at least 1% over oxygen saturation prior tothe treatment cycle.
 39. The method of claim 1, wherein the time topulmonary exacerbation in the patient is about 20 days or longer. 40.The method of claim 1, wherein the time to rescue treatment is about 20days or longer.
 41. The method of claim 1, wherein the lipid to amikacinweight ratio of the nebulized liposomal amikacin formulation is fromabout 0.3 to about 1.0 by weight. 42.-43. (canceled)
 44. The method ofclaim 1, wherein the phospholipid is selected from the group consistingof egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), eggphosphatidylinositol (EPI), egg phosphatidylserine (EPS),phosphatidylethanolamine (EPE), phosphatidic acid (EPA), soyphosphatidylcholine (SPC), soy phosphatidylglycerol (SPG), soyphosphatidylserine (SPS), soy phosphatidylinositol (SPI), soyphosphatidylethanolamine (SPE), soy phosphatidic acid (SPA),hydrogenated egg phosphatidylcholine (HEPC), hydrogenated eggphosphatidylglycerol (HEPG), hydrogenated egg phosphatidylinositol(HEPI), hydrogenated egg phosphatidylserine (HEPS), hydrogenatedphosphatidylethanolamine (HEPE), hydrogenated phosphatidic acid (HEPA),hydrogenated soy phosphatidylcholine (HSPC), hydrogenated soyphosphatidylglycerol (HSPG), hydrogenated soy phosphatidylserine (HSPS),hydrogenated soy phosphatidylinositol (HSPI), hydrogenated soyphosphatidylethanolamine (HSPE), hydrogenated soy phosphatidic acid(HSPA), dipalmitoylphosphatidylcholine (DPPC),dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol(DMPG), dipalmitoylphosphatidylglycerol (DPPG),distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol(DSPG), dioleylphosphatidylethanolamine (DOPE),palmitoylstearoylphosphatidylcholine (PSPC),palmitoylstearolphosphatidylglycerol (PSPG),mono-oleoyl-phosphatidylethanolamine (MOPE), dilauroylethylphosphocholine (DLEP), dimyristoyl ethylphosphocholine (DMEP),dipalmitoyl ethylphosphocholine (DPEP) and distearoylethylphosphocholine (DSEP),N-(2,3-di-(9-(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammoniumchloride (DOTMA), 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane(DOTAP), phosphatidylglycerols (PGs), phosphatidic acids (PAs),phosphatidylinositols (PIs), phosphatidyl serines (PSs),distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylacid(DMPA), dipalmitoylphosphatidylacid (DPPA), distearoylphosphatidylacid(DSPA), dimyristoylphosphatidylinositol (DMPI),dipalmitoylphosphatidylinositol (DPPI), distearoylphospatidylinositol(DSPI), dimyristoylphosphatidylserine (DMPS),dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine(DSPS), and mixtures thereof.
 45. (canceled)
 46. The method of claim 1wherein the phospholipid is a phosphatidylcholine. 47.-50. (canceled)